Method to determine predisposition to hypertension

ABSTRACT

The association of the molecular variant G-6A of the angiotensinogen gene with human hypertension is disclosed. The determination of this association enables the screening of persons to identify those who have a predisposition to high blood pressure.

This invention was made with Government support under Grant Nos. HL24855and HL45325, awarded by the National Institutes of Health, Bethesda, Md.The United States Government has certain rights in the invention.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a continuation of application Ser. No.08/319,545 filed on Oct. 7, 1994, now U.S. Pat. No. 5,763,168, which isa continuation-in-part of application Ser. No. 07/952,442, filed on Sep.30, 1992, now U.S. Pat. No. 5,374,525.

BACKGROUND OF THE INVENTION

The present invention relates to molecular variants of theangiotensinogen gene. The present invention further relates to thediagnosis of these variants for the determination of a predisposition tohypertension and the management of hypertension.

The publications and other materials used herein to illuminate thebackground of the invention, or provide additional details respectingthe practice, are incorporated by reference and for convenience arerespectively grouped in the appended List of References.

Hypertension is a leading cause of human cardiovascular morbidity andmortality, with a prevalence rate of 25-30% of the adult Caucasianpopulation of the United States (JNC Report, 1985). The primarydeterminants of essential hypertension, which represents 95% of thehypertensive population, have not been elucidated in spite of numerousinvestigations undertaken to clarify the various mechanisms involved inthe regulation of blood pressure. Studies of large populations of bothtwins and adoptive siblings, in providing concordant evidence for stronggenetic components in the regulation of blood pressure (Ward, 1990),have suggested that molecular determinants contribute to thepathogenesis of hypertension. However, there is no information about thegenes actually involved, about the importance of their respectiveeffects on blood pressure, or about their interactions with each otherand the environment.

Among a number of factors for regulating blood pressure, therenin-angiotensin system plays an important role in salt-waterhomeostasis and the maintenance of vascular tone; stimulation orinhibition of this system respectively raises or lowers blood pressure(Hall and Guyton, 1990), and may be involved in the etiology ofhypertension. The renin-angiotensin system includes the enzymes reninand angiotensin-converting enzyme and the protein angiotensinogen (AGT).Angiotensinogen is the specific substrate of renin, an aspartylprotease. The structure of the AGT gene has been characterized (Guillardet al., 1989; Fukamizu et al., 1990).

The human AGT gene contains five exons and four introns which span 13Kb. The first exon (37 bp) codes for the 5' untranslated region of themRNA. The second exon codes for the signal peptide and the first 252amino acids of the mature protein. Exons 3 and 4 are shorter and codefor 90 and 48 amino acids, respectively. Exon 5 contains a short codingsequence (62 amino acids) and the 3'-untranslated region.

Plasma angiotensinogen is primarily synthesized in the liver under thepositive control of estrogens, glucocorticoids, thyroid hormones, andangiotensin II (Clauser et al., 1989) and is secreted through theconstitutive pathway. Cleavage of the amino-terminal segment ofangiotensinogen by resin releases a decapeptide prohormone,angiotensin-I, which is further processed to the active octapeptideangiotensin II by the dipeptidyl carboxypeptidase angiotensin-convertingenzyme (ACE). Cleavage of angiotensinogen by renin is the rate-limitingstep in the activation of the renin-angiotensin system (Sealey andLaragh, 1990). Several observations point to a direct relationshipbetween plasma angiotensinogen concentration and blood pressure; (1) adirect positive correlation (Walker et al., 1979); (2) highconcentrations of plasma angiotensinogen in hypertensive subjects and inthe offspring of hypertensive parents compared to normotensives (Fasolaet al., 1968); (3) association of increased plasma angiotensinogen withhigher blood pressure in offspring with contrasted parentalpredisposition to hypertension (Watt et al., 1992); (4) decreased orincreased blood pressure following administration of angiotensinogenantibodies (Gardes et al., 1982) or injection of angiotensinogen (Menardet al., 1991); (5) expression of the angiotensinogen gene in tissuesdirectly involved in blood pressure regulation (Campbell and Habener,1986); and (6) elevation of blood pressure in transgenic animalsoverexpressing angiotensinogen (Ohkubo et al., 1990; Kimura et al.,1992).

Recent studies have indicated that renin and ACE are excellentcandidates for association with hypertension. The human renin gene is anattractive candidate in the etiology of essential hypertension: (1)renin is the limiting enzyme in the biosynthetic cascade leading to thepotent vasoactive hormone, angiotensin II; (2) an increase in reninproduction can generate a major increase in blood pressure, asillustrated by renin-secreting tumors and renal artery stenosis; (3)blockade of the renin-angiotensin system is highly effective in thetreatment of essential hypertension as illustrated by angiotensinI-converting enzyme inhibitors; (4) genetic studies have shown thatrenin is associated with the development of hypertension in some ratstrains (Rapp et al. 1989; Kurtz et al. 1990); (5) transgenic animalsbearing either a foreign renin gene alone (Mullins et al. 1990) or incombination with the angiotensinogen gene (Ohkubo et al. 1990) developprecocious and severe hypertension.

The human ACE gene is also an attractive candidate in the etiology ofessential hypertension. ACE inhibitors constitute an important andeffective therapeutic approach in the control of human hypertension(Sassaho et al. 1987) and can prevent the appearance of hypertension inthe spontaneously hypertensive rat (SHR) (Harrap et al., 1990).Recently, interest in ACE has been heightened by the demonstration oflinkage between hypertension and a chromosomal region including the ACElocus found in the stroke-prone SHR (Hilbert et al., 1991; Jacob et al.,1991).

The etiological heterogeneity and multifactorial determination whichcharacterize diseases as common as hypertension expose the limitationsof the classical genetic arsenal. Definition of pheno-type, model ofinheritance, optimal familial structures, and candidate-gene versusgeneral-linkage approaches impose critical strategic choices (Lander andBotstein, 1986; White and Lalouel, 1987; Lander and Botstein, 1989;Lalouel, 1990, 1990; Lathrop and Lalouel, 1991). Analysis by classicallikelihood ratio methods in pedigrees is problematic due to the likelyheterogeneity and the unknown mode of inheritance of hypertension. Whilesuch approaches have some power to detect linkage, their power toexclude linkage appears limited. Alternatively, linkage analysis inaffected sib pairs is a robust method which can accommodateheterogeneity and incomplete penetrance, does not require any a prioriformulation of the mode of inheritance of the trait and can be used toplace upper limits on the potential magnitude of effects exerted on atrait by inheritance at a single locus. (Blackwelder and Elston, 1985;Suarez and Van Eerdewegh, 1984).

It was an object of the present invention to determine a geneticassociation with essential hypertension. It was a further object toutilize such an association to identify persons who may be predisposedto hypertension leading to better management of the disease.

SUMMARY OF THE INVENTION

The present invention relates to the identification of a molecular basisof human hypertension. More specifically, the present invention hasidentified that angiotensinogen (AGT) is involved in the pathogenesis ofhypertension. Molecular variants of the AGT gene contribute to anindividual's susceptibility to the development of hypertension. Theanalysis of the AGT gene will identify subjects with a geneticpredisposition to develop essential hypertension or pregnancy-inducedhypertension. The management of hypertension in these subjects couldthen be more specifically managed, e.g., by dietary sodium restriction,by carefully monitoring blood pressure and treating with conventionaldrugs, by the administration of renin inhibitors or by theadministration of drugs to inhibit the synthesis of AGT. The analysis ofthe AGT gene is performed by comparing the DNA sequence of anindividual's AGT gene with the DNA sequence of the native, non-variantAGT gene.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Genotyping with a dinucleotide repeat at the angiotensinogenlocus in hypertensive sibships. Representative genotypes for the AGT GTrepeat are shown. Familial relationships in six hypertensive sibshipsare shown at the top of the figure. Genomic DNA of each individual wasamplified with primers for the GT repeat at the AGT locus, fractionatedvia electrophoresis and subjected to autoradiography as described in theExamples; results for each individual are shown below, with assignedgenotypes for each individual indicated at the bottom of the figure.Each allele characteristically shows a single dark band and a fainterband which is shorter by 2 base pairs; alleles have been scoredaccording to the darker band.

FIGS. 2A and 2B. Identification and DNA sequence analysis of variants inthe angiotensinogen gene. Segments of the angiotensinogen gene wereamplified and fractionated via electrophoresis on non-denaturing gels asdescribed in the Examples. Autoradiograms showing the products ofamplification of different hypertensive subjects are shown.

FIG. 2A shows products of an individual homozygous for the T174M variant(indicated by arrow) and two subjects homozygous for T174. Sequences ofthese different products were determined as described in the Examplesand are shown below, with the T174 sequence shown above thecorresponding T174M sequence (sequences are of the anti-sense strandwith 5' to 3' orientation from left to right). The nucleotidesubstitution distinguishing the variants is indicated by *.

FIG. 2B shows products of two individuals homozygous for M235T(indicated by arrows) and three subjects homozygous for M235. Thecorresponding sequences are again shown below.

FIG. 3. Map of the human genomic angiotensinogen gene and location ofidentified variants. Exons of the angiotensinogen gene are representedby open boxes at the top of the figure. An expanded view of the 5'flanking region of the gene is shown below, with the location oftranscriptional regulatory sequences indicated by filled boxes: CAT,TATA and RNA polymerase III (RPP) promoter elements; hormone responsiveelements for glucocorticoid (GRE, P.GRE: putative GRE), estrogen (ERE),or thyroid hormone (TRE); hepatic specific element (HSE); acute phaseresponse element (APRE); putative enhancer element (ENH). The locationsof sequence variants identified in hypertensive subjects are indicatedby numbered arrows; exact location and nature of each variant isindicated in Table 2 below.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to the determination that molecularvariants of the angiotensinogen (AGT) gene are involved in thepathogenesis of hypertension. The present invention has surprisinglyfound that molecular variants of the AGT gene contribute to thedevelopment of hypertension in humans. The present invention is furtherdirected to methods of screening humans for the presence of AGT genevariants which are associated with the predisposition of humans todevelop essential hypertension or pregnancy-induced hypertension. Sincea predisposition to hypertension can now be established by screening formolecular variants of the AGT gene, individuals at risk can be moreclosely monitored and treated before the disease becomes serious.

Essential hypertension is one of the leading causes of humancardiovascular morbidity and mortality. Epidemiological studies of bloodpressure in related individuals suggest a genetic heritability around30% (Ward, 1990) The continuous, unimodal distribution of blood pressurein the general population as well as in the offspring of hypertensiveparents (Hamilton et al., 1954) supports the hypothesis that severalgenes are involved in this genetic predisposition. However, there is noinformation about the genes actually involved, about the importance oftheir respective effects on blood pressure, or their interactions witheach other and the environment.

Genetic studies in animal models of hypertension have suggested aninvolvement of the two key enzymes of this system in the genesis of highblood pressure, renin (Rapp et al., 1989; Mullins et al., 1990; Kurtz etal., 1990), and angiotensin converting enzyme through linkage to anearby marker (Hilbert et al., 1991; Jacob et al., 1991; Deng and Rapp,1992). The purpose of the present invention was to identify anassociation with hypertension. It was unexpectedly found that neitherrenin nor angiotensin-converting enzyme is associated with humanhypertension. Instead, it was found that the angiotensinogen gene isinvolved in the pathogenesis of essential hypertension. The followingwere found: (1) genetic linkage between essential hypertension and AGTin affected siblings; (2) association between hypertension and certainmolecular variants of AGT as revealed by a comparison between cases andcontrols; (3) increased concentrations of plasma angiotensinogen inhypertensive subjects who carry a common variant of AGT stronglyassociated with hypertension; (4) persons with the most common AGT genevariant exhibited not only raised levels of plasma angiotensinogen butalso higher blood pressure; and (5) the most common AGT gene variant wasfound to be statistically increased in women presenting preeclampsiaduring pregnancy, a condition occurring in 5-10% of all pregnancies.

The association between renin, ACE or AGT and essential hypertension wasstudied using the affected sib pair method (Bishop and Williamson, 1990)on populations from Salt Lake City, Utah and Paris, France, as describedin further detail in the Examples. Only an association between the AGTgene and hypertension was found. The AGT gene was examined in personswith hypertension, and at least 15 variants have been identified. Noneof these variants occur in the region of the AGT protein cleaved byeither renin or ACE. The identification of the AGT gene as beingassociated with essential hypertension was confirmed in a populationstudy of healthy subjects and in women presenting preeclampsia duringpregnancy.

Although molecular variants of the AGT gene have been established aspredisposing a person to hypertension, it is not possible to determineat this time whether the observed molecular variants of AGT directlyaffect function or whether they serve as markers for functional variantsthat have escaped identification by the molecular screening method used.When the sequence of human angiotensinogen is compared to that of ratangiotensinogen, and to other serine protease inhibitors (serpins) suchas antithrombin-III and alpha-1-antitrypsin, the AGT gene variants M235Tand T174M appear to occur in regions with little conservation (Carrelland Boswell, 1986). By contrast, the variant Y248C, which was observedin the heterozygote state in only one pair of hypertensive siblings,constitutes a non-conservative substitution in a region well conservedamong serpins. In addition to this predisposition encoded by commonvariants, rare variants such as Y248C and V388M have the potential toimpart predispositions with unique clinical courses and severities.

As used herein, AGT gene variants are expressed either at the amino acidlevel, e.g., M235T in which the variant protein contains threonine atamino acid residue 235 instead of methionine, or at the nucleotidelevel, e.g., C-532T in which the variant gene contains thymidine atnucleotide-532 of the 5' sequence instead of cytosine of the nativegene. Several mutations are set forth in Table 2.

When hypertensive siblings were stratified according to genotypes atresidue 235, higher plasma concentrations of angiotensinogen wereobserved among carriers of M235T (F₂,313 =14.9, p<0.0001). Again, thisresult was observed independently in each sample. A correlation betweenplasma angiotensinogen concentration and blood pressure has already beenobserved (Walker et al., 1979). Taken together, these observationssuggest a direct involvement of plasma angiotensinogen in thepathogenesis of essential hypertension. This conclusion is furtherstrengthened by finding that the M235T variant was significantlyassociated not only with raised plasma angiotensinogen concentrationsbut also with increased blood pressure. See Example 8, below. Thepresent invention is corroborated by two additional findings: (1) plasmaangiotensinogen was higher in hypertensive subjects and in the offspringof hypertensive parents than in normotensives (Fasola et al., 1968); and(2) in the Four-Corners study, angiotensinogen concentrations weresignificantly associated with increased blood pressure in the subsetmost likely to entail a genetic predisposition, namely the high-bloodpressure offspring of high-blood pressure parents (Watt et al., 1992).Because the plasma concentration of angiotensinogen is close to theK_(m) of the enzymatic reaction between renin and angiotensinogen (Gouldand (Green, 1971), a rise or fall in renin substrate can lead to aparallel change in the formation of angiotensin II (Cain et al., 1971;Menard and Catt, 1973; Arnal et al., 1991). Therefore, it is conceivablethat raised baseline levels could lead to mild overactivity of therenin-angiotensin system, and represent an altered homeostatic setpointin predisposed individuals. Indeed, long-term administration ofangio-tensin II at subpressor doses has been shown to elevate bloodpressure (Brown et al., 1981).

Recent studies suggest that not only plasma angiotensinogen, but alsolocal expression in specific tissues, could contribute to blood pressureregulation. Yongue et al. (1991) observed increased expression ofangiotensinogen in the anteroventral hypothalamus and in contiguousareas of the brain in SHR rats in comparison to normotensive control WKYrats, but they found no difference in liver expression. A possible roleof angiotensinogen in the central nervous system is further supported byexperimental overexpression of the AGT gene in transgenic rats: plasmaconcentrations were raised, but high blood pressure was observed only ina transgenic line displaying proper tissue-specific expression of thetransgene in the brain (Kimura et al., 1992). Furthermore, evidence forlocal synthesis of the different components of the renin angiotensinsystem in the kidney has accumulated and an alteration of the regulationof angiotensinogen expression by sodium has been observed in SHR rats(Pratt et al., 1989).

Without being bound by any theory of action, it is possible that somemolecular variants of angiotensinogen, such as those identified ortagged by the variant at residue 235 or the variant at the -6nucleotide, lead to increased plasma or tissue angiotensinogen as aresult of either increased synthetic rate, altered reaction constantswith renin, or increased residence time through complex formation withself or with other extracellular proteins. This could lead to a smallincrease in baseline or in reactive production of angiotensin II,accounting for a slight overreactivity of the renin-angiotensin systemin response to sodium and environmental stressors. Over decades, this inturn could promote sodium retention as a result of chronic stimulationof aldosterone secretion, vascular hypertrophy and increased peripheralvascular resistance as a result of chronic elevation of angiotensin IIformation, or abnormal stimulation of the sympathetic nervous systemmediated by enhanced production of angiotensin II in relevant areas ofthe brain.

The identification of the association between the AGT gene andhypertension permits the screening of individuals to determine apredisposition to hypertension. Those individuals who are identified atrisk for the development of the disease may benefit from dietary sodiumrestriction, can have their blood pressure more closely monitored and betreated at an earlier time in the course of the disease. Such bloodpressure monitoring and treatment may be performed using conventionaltechniques well known in the art.

To identify persons having a predisposition to hypertension, the AGTalleles are screened for mutations. Plasma angiotensirogen levels ofpersons carrying variants of the AGT gene are then examined to identifythose at risk. Any human tissue can be used for testing the DNA. Mostsimply, blood can be drawn and DNA extracted from the cells of theblood. The AGT alleles are screened for mutations either directly orafter cloning the alleles.

The alleles of the AGT gene in an individual to be tested are clonedusing conventional techniques. For example, a blood sample is obtainedfrom the individual. The genomic DNA isolated from the cells in thissample is partially digested to an average fragment size ofapproximately 20 kb. Fragments in the range from 18-21 kb are isolated.The resulting fragments are ligated into an appropriate vector. Thesequences are then analyzed as described herein.

Alternatively, polymerase chain reactions (PCRs) are performed withprimer pairs for the 5' region or the exons of the AGT gene. Examples ofsuch primer pairs are set forth in Table 1. PCRs can also be performedwith primer pairs based on any sequence of the normal AGT gene. Forexample, primer pairs for the large intron can be prepared and utilized.Finally, PCR can also be performed on the mRNA. The amplified productsare then analyzed as described herein.

                                      TABLE 1                                     __________________________________________________________________________    Primers Used for the Detection                                                  of Molecular Variants of AGT                                                Location                                                                           Primer 1 (SEQ ID NO:)                                                                           Primer 2 (SEQ ID NO:)                                  __________________________________________________________________________    5'   ACCATTTGCAATTTGTACAGC                                                                        (1)                                                                              GCCCGCTCATGGGATGTG                                                                          (2)                                         - 5'   AAGACTCTCCCCTGCCCCTCT  (3) GAAGTCTTAGTGATCGATGCAG (4)                  - 5+ +Exl AGAGGTCCCAGCGTGAGTGT  (5)    AGACCAGAAGGAGCTGAGGG (6)                                                      - Ex2               GTTAATAACCAC                                            CTTTCACCCTT (7)    GCAGGTATGAAGGTG                                            GGGTC   (8)                              - Ex2               AGGCCAATGCCGGGAAGCCC    (9) ATCAGCCCTGCCCTGGGCCA                                               (10)                                     - Ex2               GATGCGCACAAGGTCCTGTC   (11)   GCCAGCAGAGAGGTTTGCCT                                               (12)                                   - Ex3               TCCCTCCCTGTCTCCTGTCT   (13) TCAGGAGAGTGTGGCTCCCA                                               (14)                                     - Ex4               TGGAGCCTTCCTAACTGTGC   (15)   AGACACAGGCTCACACATAC                                               (16)                                   - Ex5               GTCACCCATGCGCCCTCAGA   (17)   GTGTTCTGGGGCCCTGGCCT                                               (15)                                __________________________________________________________________________

The alleles are tested for the presence of nucleic acid sequencedifferences from the normal allele by determining the nucleotidesequence of the cloned allele or amplified fragment and comparing it tothe nucleotide sequence of the normal allele. Alternatively, there aresix well known methods for a more complete, yet still indirect, test forconfirming the presence of a predisposing allele: 1) single strandedconformation analysis (SSCA) (Orita et al., 1989); 2) denaturinggradient gel electrophoresis (DGGE) (Wartell et al., 1990; Sheffield etal., 1989); 3) RNase protection assays (Finkelstein et al., 1990;Kinszler et al., 1991); 4) allele-specific oligonucleotides (ASOs)(Conner et al., 1983); 5) the use of proteins which recognize nucleotidemismatches, such as the E. coli mutS protein (Modrich, 1991); and, 6)allele-specific PCR (Rano & Kidd, 1989). For allele-specific PCR,primers are used which hybridize at their 3' ends to a particular AGTmutation. If the particular AGT mutation is not present, anamplification product is not observed. Amplification Refractory MutationSystem (ARMS) can also be used, as disclosed in European PatentApplication Publication No. 0332435 and in Newton et al., 1989.

In the first three methods (SSCA, DGGE and RNase protection assay), anew electrophoretic band appears. SSCA detects a band which migratesdifferentially because the sequence change causes a difference insingle-strand, intramolecular base pairing. RNase protection involvescleavage of the mutant polynucleotide into two or more smallerfragments. DGGE detects differences in migration rates of mutantsequences compared to wild-type sequences, using a denaturing gradientgel. In an allele-specific oligonucleotide assay, an oligonucleotide isdesigned which detects a specific sequence, and the assay is performedby detecting the presence or absence of a hybridization signal. In themutS assay, the protein binds only to sequences that contain anucleotide mismatch in a heteroduplex between mutant and wild-typesequences.

Mismatches, according to the present invention, are hybridized nucleicacid duplexes in which the two strands are not 100% complementary. Lackof total homology may be due to deletions, insertions, inversions orsubstitutions. Mismatch detection can be used to detect point mutationsin the gene or in its mRNA product. While these techniques are lesssensitive than sequencing, they are simpler to perform on a large numberof samples. An example of a mismatch cleavage technique is the RNaseprotection method. In the practice of the present invention, the methodinvolves the use of a labeled riboprobe which is complementary to thehuman wild-type AGT gene coding sequence. The riboprobe and either mRNAor DNA isolated from the tumor tissue are annealed (hybridized) togetherand subsequently digested with the enzyme RNase A which is able todetect some mismatches in a duplex RNA structure. If a mismatch isdetected by RNase A, it cleaves at the site of the mismatch. Thus, whenthe annealed RNA preparation is separated on an electrophoretic gelmatrix, if a mismatch has been detected and cleaved by RNase A, an RNAproduct will be seen which is smaller than the full length duplex RNAfor the riboprobe and the mRNA or DNA. The riboprobe need not be thefull length of the AGT mRNA or gene but can be a segment of either. Ifthe riboprobe comprises only a segment of the AGT mRNA or gene, it willbe desirable to use a number of these probes to screen the whole mRNAsequence for mismatches.

In similar fashion, DNA probes can be used to detect mismatches, throughenzymatic or chemical cleavage. See, e.g., Cotton et al., 1988; Shenk etal., 1975; Novack et al., 1986. Alternatively, mismatches can bedetected by shifts in the electrophoretic mobility of mismatchedduplexes relative to matched duplexes. See, e.g., Cariello, 1988. Witheither riboprobes or DNA probes, the cellular mRNA or DNA which mightcontain a mutation can be amplified using PCR before hybridization.Changes in DNA of the AGT gene can also be detected using Southernhybridization, especially if the changes are gross rearrangements, suchas deletions and insertions.

DNA sequences of the AGT gene which have been amplified by use of PCRmay also be screened using allele-specific probes. These probes arenucleic acid oligomers, each of which contains a region of the AGT genesequence harboring a known mutation. For example, one oligomer may beabout 30 nucleotides in length, corresponding to a portion of the AGTgene sequence. By use of a battery of such allele-specific probes, PCRamplification products can be screened to identify the presence of apreviously identified mutation in the AGT gene. Hybridization ofallele-specific probes with amplified AGT sequences can be performed,for example, on a nylon filter. Hybridization to a particular probeunder stringent hybridization conditions indicates the presence of thesame mutation in the DNA sample as in the allele-specific probe.

Mutations falling outside the coding region of AGT can be detected byexamining the noncoding regions, such as introns and regulatorysequences near or within the AGT gene. An early indication thatmutations in noncoding regions are important may come from Northern blotexperiments that reveal messenger RNA molecules of abnormal size orabundance in hypertensive patients as compared to control individuals.

Alteration of AGT mRNA expression can be detected by any techniquesknown in the art. These include Northern blot analysis, PCRamplification and RNase protection. Diminished mRNA expression indicatesan alteration of the wild-type AGT gene. Alteration of wild-type AGTgenes can also be detected by screening for alteration of wild-typeangiotensinogen. For example, monoclonal antibodies immunoreactive withangiotensinogen can be used to screen a tissue. Lack of cognate antigenwould indicate a AGT gene mutation. Antibodies specific for products ofmutant alleles could also be used to detect mutant AGT gene product.Such immunological assays can be done in any convenient formats known inthe art. These include Western blots, immunohistochemical assays andELISA assays. Any means for detecting an altered angiotensinogen can beused to detect alteration of wild-type AGT genes. Finding a mutant AGTgene product indicates alteration of a wild-type AGT gene.

Further details of a suitable PCR method are set forth in the Examples.The AGT alleles can be screened for the variants set forth in Table 2,as well as other variants using these techniques or those techniquesknown in the art.

                                      TABLE 2                                     __________________________________________________________________________    Molecular Variants in the Angiotensinogen Gene                                                               Allele Frequency                                              Substitution    Salt Lake City.sup.1                                                                 Paris.sup.2                             Variant                                                                           Location                                                                           Position.sup.3                                                                      Nucleotide                                                                             Amino Acid                                                                           H/C    H/C                                     __________________________________________________________________________     1  5'   -532  C → T    .13/.12                                                                               .11/n.d.                                  2 5' -386 G → A  .04/.04  .02/n.d.                                     3 5' -218 G → A  .11/.10  .08/n.d.                                     4 5'  -18 C → T  .13/.13  .19/n.d.                                     5 5' -6 and -20 G → A and A → C  .19/.14  .18/n.d.                                                   6 Ex 1  +10 C → T untransla                                          ted 1 ind/0       0/n.d.                   7 Ex 2 +521 C → T T → M (174) .sup. .18/.08.sup.4 .sup.                                            .17/.08.sup.4                              8 Ex 2 +597 T → C P → P (199) 1 ind/0       0/n.d.                                                   9 Ex 2 +704 T → C M                                                  → T (235) .sup. .49/.36.sup.5                                           .sup. .52/.38.sup.4                      10 Ex 2 +743 A → G Y → C (248) 1 ind/0       0/n.d.                                                  11 Ex 3 +813 C → T N                                                  → N (271) 1 ind/0    0/0                                                12 Ex 3 +1017  G → A L                                                → L (339) .05/.08  .06/n.d.                                             13 Int 3 .sup.  -13.sup.6 A                                                  → G  .07/.11  .08/n.d.                                                  .sup. 14.sup.7 Ex 4 +1075  C                                                 → A L → M (359)                                                 .005/.01  n.d./n.d.                       15 Ex 4 +1162  G → A V → M (388) 0/0    0/1 ind               __________________________________________________________________________     .sup.1 Salt Lake City: 90 controls, 36 index patients from most severely      affected pairs.                                                               .sup.2 Paris: 98 controls, 43 index patients from most severely affected      pairs.                                                                        .sup.3 Position is with reference to transcription start site.                .sup.4 p < 0.01                                                               .sup.5 p < 0.05                                                               .sup.6 Position relative to beginning of exon 4.                              .sup.7 Variant previously described suppressing a PstI site (Kunapuli and     Kumer, 1986).                                                                 H/C  Hypertensive/Control                                                     n.d.  not done.                                                               1 ind  1 individual detected with the corresponding molecular variant.   

The present invention is further detailed in the following Examples,which are offered by way of illustration and are not intended to limitthe invention in any manner. Standard techniques well known in the artor the techniques specifically described below are utilized.

EXAMPLE 1 Selection of Sibships with Multiple Hypertensive Subjects

A. Salt Lake City

Families with two or more hypertensive siblings were characterized andsampled from "Health Family Tree" questionnaires collected from theparents of 40,000 high school students in Utah. The characteristics ofthis population-based selection of hypertensive sibships have beendescribed previously (Williams et al., 1988). For purposes of thepresent study, affection status was defined as a diagnosis ofhypertension requiring treatment with antihypertensive medication priorto age 60, and the absence of diabetes mellitus or renal insufficiency;the study sample comprises 309 siblings (165 women, 144 men). All butthree sibling pairs were Caucasians (one was Asian, two Hispanic) andtheir relevant clinical characteristics are indicated in Table 3. The132 affected sibships are composed of 102 duos, 20 trios, sevenquartets, one quintet, and two sextets of hypertensive siblings.

B. Paris

The selection of hypertensive families with a high prevalence ofessential hypertension was conducted through ascertainment ofhypertensive probands referred to the Hypertension Clinic of theBroussais Hospital in Paris, as previously described (Corvol et al.,1989). The 83 French sibships were collected through index patients whosatisfied the following criteria: (1) onset of hypertension before age60; (2) established hypertension defined either by chronically treatedhypertension (n=156) or by a diastolic blood pressure greater than 95mmHg at two consecutive visits for those without antihypertensivetreatment (n=34, mean diastolic blood pressure=103.8±13.1 mmHg); (3)absence of secondary hypertension, established by extensive inpatientwork-up when clinically indicated; and (4) familial history of earlyonset (before age 60) of hypertension in at least one parent and onesibling. Patients with exogenous factors that could influence bloodpressure were eliminated, in particular those with alcohol intake ofmore than four drinks per day or women taking oral contraceptives. Otherexclusion criteria were a body mass index (BMI=weight/height²) greaterthan 30 kg/m², the presence of diabetes mellitus, or renalinsufficiency; the total sample consisted of 83 hypertensive sibshipswith 62 duos, 19 trios, 1 quartet and 1 quintet. All subjects wereCaucasians and their relevant clinical characteristics are summarized inTable 3, below.

C. Controls

In Salt Lake City 140 controls were defined as the grandparents of theUtah families included in the CEPH data base (Centre d'Etude duPolymorphisme Humain), a random panel of healthy families with largesibship size that serves as reference for linkage studies (Dausset etal., 1990). The French controls were 98 healthy normotensive subjectswho had been selected in the context of a previous case-control study(Soubrier et al., 1990). Both samples included only Caucasians.

                  TABLE 3                                                         ______________________________________                                        Clinical Characteristics                                                        of the Hypertensive Siblings                                                             Salt Lake City Paris                                             ______________________________________                                        Sibships     132     (244)      83   (135)                                      (pairs)                                                                       Subjects (m/f) 309 (144/165) 190 (99/91)                                      Age (years) 49.4 (±7.4) 52.3 (±9.9)                                     Age dx (years) 39.4 (±9.6) 40.4 (±11.7)                                 SBP (mmHg) 127.8 (±15.6) 156.0 (±21.5)                                  DBP (mmHg) 80.0 (±9.9) 98.2 (±12.6)                                     Rx (%) 309 (100%) 158 (82%)                                                   B.M.I. (Kg/m.sup.2) 29.7 (±5.5) 24.9 (±3.0)                           ______________________________________                                         Age dx: Age of diagnosis                                                      SBP and DBP: Systolic and Diastolic Blood Pressure                            B.M.I.: Body Mass Index                                                       Unless otherwise stated, values are indicated as mean ± 1 S.D.        

EXAMPLE 2 General Methods for Analysis of Linkage With Renin

A. Experimental Protocols

The experimental protocols using the French populations were conductedas previously described (Soubrier et al. 1990). Briefly, two probes wereused to detect the diallelic RFLPs of three restriction enzymes. A1.1-kb human renin cDNA fragment (Soubrier et al. 1983) was used todetect the HindIII polymorphism and a 307-bp genomic DNA fragmentlocated in the 5' region of the renin gene (Soubrier et al. 1986) wasused to detect the TaqI and HinfI polymorphisms. These two probes werelabeled at high specific activity (4×10⁹ to 8×10⁹ cpm/mg) with therandom primer labelling method (Feinberg and Vogelstein 1983).

Human genomic DNA was digested by TaqI, HinfI, or HindIII (New EnglandBiolabs, Beverly, Mass.) and subjected to electrophoresis through anagarose gel (0.7% or 1.2%). After alkaline transfer to a nylon membrane(Hybond-N+, Amersham), hybridization to the corresponding probe, washingunder high stringency conditions, and autoradiography, each restrictionendonuclease detected the following biallelic RFLPs: 11- and 9.8-kballeles (TaqI), 1.4- and 1.3-kb alleles (HinfI), and 9.0- and 6.2-kballeles (HindIII). These polymorphisms and their frequencies were inaccordance with those previously described (Frossard et al., 1986 a,b;Masharani, 1989; Naftilan et al., 1989).

B. Analysis of RFLP Frequencies

For each RFLP, allele frequencies were determined from the genotypefrequencies that had been previously established in 120 normotensivesand 102 hypertensives (Soubrier et al. 199). These frequencies satisfiedthe Hardy-Weinberg equilibrium. The informativeness of each biallelicRFLP, estimated by the polymorphism information content (PIC), wasrespectively 0.16 (TaqI), 0.33 (HindIII), and 0.27 (HinfI). In spite oflinkage disequilibriums between the HinfI-HindIII and HinfI-TaqIpolymorphisms, the combination of the three RFLPs led to a markedimprovement in the marker's informativeness (PIC=0.65), corresponding to70% of heterozygosity.

C. Construction of Haplotypes

The haplotypes were deduced from the combination of the three diallelicRFLPs. By the presence or absence of each restriction enzyme site, itwas possible to define 8 (2³) different haplotypes and 27 (3³)genotypes. The haplotype frequencies have been previously estimated on ahypertensive population (Soubrier et al. 1990), with a maximumlikelihood technique according to Hill's method (Hill 1975). Thesehaplotypes were used as a new multiallelic system in which each allelecorresponded to one haplotype, numbered by its order frequency. Thesefrequencies enabled us to compute the expected values of the number ofalleles shared by a sibship under the hypothesis of an independentsegregation of the renin gene marker and hypertension.

D. Comparison of Sib Genotypes

In 12 sibships, it was not possible to determine with certainty eachhaplotype--the presence of double or triple heterozygosity in therestriction enzyme sites--in spite of the analysis of other members ofthe same family. In these cases, the relative different parental matingtype probabilities were calculated according to the haplotypefrequencies. Then, the probabilities of the genotypes of each sib pairwere deduced conditional to each parental mating type. For each sibship,the concordance between sibs was calculated as the mean of all possibleconcordances according to their relative probabilities.

Because of the absence of one or two parental genotypes in 40 of the 57sibships, and of the absence of complete heterozygosity of the reninmarker, the alleles shared in common by one sib pair were assumed to beidentical by state (ibs), rather than identical by descent. Theconcordance between the sib genotypes could be total (ibs=1), partial(ibs=1/2), or absent (ibs=0). Under the null hypothesis of no linkage,the mean number of identical market alleles shared by a set of sib pairs(and its variance) is not affected by whether or not some of the sibpairs belong to the same sibship (Suarez et al. 1983, Blackwelder andElston 1985). Thus, the renin genotypes were compared for each sib pairand all the information contained in each sibship was taken into accountby adding the concordances between all different sib pairs.

E. Comparison of the Expected Concordance Values

The expected proportions of alleles shared by both sibs were computedaccording to Lange (1986). This statistical method first calculates theprobabilities of the different possible parental mating types takinginto account the allelic frequencies and then the expected probabilitiesof total, half, or null concordance between sibs. It is thus possible tocalculate the mean and the variance of the expected concordance fordifferent sibship sizes under the null hypothesis of no linkage. Thefinal t statistic is a one-sided Student's test adding the contributionsof the different sibships.

Taking into account the possible bias in ascertainment of the size ofthe sibships, several authors have proposed different weights (w) tomaximize the power of this statistic. In addition to w₁ =1, we tested w₂=1/Var(Z_(s))^(1/2) (Suarex et al. 1983; Motro and Thompson 1985) and w₃=(s-1)^(1/2) /Var(Z_(s))^(1/2) (Hodge 1984), where s represents the sizeof the affected sibship and Z, the statistic reflecting the allelicconcordance for each sibship size (Lange 1986).

EXAMPLE 3 General Methods for Analysis of Linkage with ACE

A. Genotypes

(1) The hGH-A1819 primers were designed from the published sequenceflanking the eighteenth and nineteenth Alu elements of the hGH gene(Chen et al., 1989): 5'-ACTGCACTCCAGCCTCGGCAG-3' (SEQ ID NO:19),5'-ACAAAAGTCCTTCTCCAGAGCA-3' (SEQ ID NO:20). Polymerase chain reactions(PCR) were performed using 100 ng of genomic DNA in a total volume of 20ml containing 1×PCR Buffer (Cetus), 125 mM dNTPs, 150 pmol primers, 2mCia 32P-dCTP. After an initial denaturation step (4 min at 94° C.),each of the 30 cycles consisted of 1 min at 94° C., 45 s at 63° C. and30 s at 72° C., followed by a final elongation step (7 min at 72° C.).PCR reactions were performed in 96-well microtitre plates, using aTechne 2 apparatus. After completion, 20 ml of formamide with 10 mM EDTAwas added to each reaction and, after denaturation of 94° C. for 5 min,1 ml of this mixture was loaded on a 6% acrylamide gel containing 30%formamide, 7M Urea, 135 mM TrisHCl, 45 mM boric acid and 2.5 mM EDTA.Gels were run at 70 W for 4 hr and were exposed 6-12 hr forautoradiography. (2) The ACE diallelic polymorphism was genotyped byenzymatic amplification of a segment in intron 16, with the 190 and 490bp alleles resolved by a 1.5% agarose gel (Rigat et al., 1992).

B. Genetic Mapping

The chromosome 17 markers used in the genetic map were developed in theDepartment of Human Genetics of the University of Utah (Nakamura et al.,1988). The pairwise lod scores and recombination estimates (r) weredetermined from the analysis of 35 and 11 CEPH reference families forthe ACE and hGH markers, respectively, using LINKAGE. (Lathrop et al.,1984) No recombination between ACE and hGH was detected from thispairwise analysis. Map order and recombination estimates of thechromosome 17 markers have then been determined using the CILINKsubroutine. The placement of ACE has been determined by linkage to thisgenetic map in which the order and recombination frequencies between allother markers, including hGH, have been fixed at their maximumlikelihood values.

C. Sib Pair Analysis

All sib pairs from multiplex sibships were considered as independent,and the statistic was based on the mean number of alleles shared.(Blackwelder and Elston, 1985) In the absence of parental genotypes, thesharing of alleles was scored as i.b.s. For each sibship size, theexpectation of the mean number of alleles shared i.b.s. and its variancewere calculated as described previously. (Lance, 1986) The results showa 0.08% excess of alleles shared (95% confidence interval±6.9%). For allpairs given equal weight, the one-sided t value is 0.02 (p=0.45).Weighting the contributions of multiplex sibships according to Hodge(Hodge, 1984) gives a final t value of 0.01 (p=0.49).

EXAMPLE 4 General Methods of Analysis of Linkage With AGT

A. Genotyping GT Alleles at the AGT Locus

AGT genotypes were established by means of a highly informativedinucleotide repeat in the 3' flanking region of the AGT gene(Kotelevtsev et al., 1991). The primers used for the Paris sample wereas published (K-primers); for the genotypes characterized in Utah,primers more distant to the (GT) repeat were designed:

5'-GGTCAGGATAGATCTCAGCT-3' (SEQ ID NO:21),

5'-CACTTGCAACTCCAGGAAGACT-3' (SEQ ID NO:22)

(U-Primers), which amplify a 167-bp fragment. In both laboratories, thepolymerase chain reactions (PCR) were performed using 80 ng of genomicDNA in a total volume of 20 μl containing 50 mM KCl, 5 mM Tris-HCl,0.01% gelatin, 1.5 mmol MgCl₂, 125 μM dNTPs, 20 pmol of each unlabeledprimer, 10 pmol of one ³² P-end labeled primer and 0.5 U of Taqpolymerase (Perkin-Elmer Cetus Norwalk, Conn.). After an initialdenaturation step (4 min at 94° C.), each of the 30 cycles consisted of1 min at 94° C., 1 min at 55° C. and 1 min at 72° C. (K-primers) or 45sec at 94° C., 45 sec at 62° C. and 30 sec at 72° C. (U-primers). Aftercompletion, 20 μl of formamide with 10 mM EDTA was added to eachreaction and, after denaturation at 94° C. for 5 min, 1 μl of thismixture was loaded on a 6% acrylamide gel containing 30%, formamide, 7MUrea, 135 mM TrisHCl, 45 mM Boric Acid and 2.5 mM EDTA (pH 7.8). Gelswere run at 70 W for 4 hours and were exposed 6-12 hours forautoradiography.

Genotypes were characterized in each of the hypertensive subjects and in117 of their first-degree relatives. Allelic frequencies were evaluatedin 98 Caucasian normotensive controls from Paris, 140 Caucasiangrandparents of CEPH pedigrees from Salt Lake City, and both sets ofhypertensive index cases. At least 10 alleles were observed in each ofthe four groups, confirming the high heterozygosity (80%) of the marker.No significant difference in allelic frequencies was observed betweencontrols and hypertensives from Paris (χ² ₆ =7.7, p=0.26); frequenciesin controls were used as reference for linkage analysis in this sample.By contrast, controls and hypertensives from Salt Lake City exhibitedsignificant differences in allelic frequencies (χ² ₆ =17.1, p<0.01),primarily because the frequency of the most common allele was lower inhypertensives (0.36) than in controls (0.40); to avoid spurious bias onlinkage tests, the frequencies estimated in hypertensive index caseswere used for the analysis of the Salt Lake City sample.

B. Analysis of Linkage in Pairs of Hypertensive Siblings

Conditional independence of segregating events within sibships (Suarezand Van Eerdewegh, 1984) led to the generation of a total of 379 pairsof hypertensive siblings. Parental genotypes were determined directly orinferred from genotypes of non-hypertensive siblings in ten of theFrench sibships. In these sibships, alleles shared by siblings wereconsidered as identical by descent (i.b.d.) and the appropriatestatistical comparison employed (mean of 1.0 alleles shared per pairunder independence). In the absence of parental genotypes (all Utahsibships, 73 French sibships), alleles shared by siblings were scored isidentical by state (i.b.s) (Suarez et al., 1978; Blackwelder and Elston,1985; Lange, 1986). For each sibship size, the expectation of the meannumber of alleles shared i.b.s., and its variance, were calculatedaccording to Lange (1986). The comparison between the observed andexpected mean numbers of alleles shared by the pairs of siblings ofevery sibship yielded a one-sided Student t-test. The contribution ofsibships of each size was weighted according to Hodge (Hodge, 1984).Predefined partitions of the data were examined sequentially so as toprovide a parsimonious management of the degrees of freedom associatedwith multiple comparisons.

C. Search for Molecular Variants

Enzymatic Amplification of Segments of the Angiotensinogen Gene

From the known genomic structure of the human angiotensinogen gene(Gaillard et al., 1989), ten different sets of oligonucleotides(Table 1) were designed to cover the 5' region containing the mainregulatory elements and the five exons of the gene. They were chosen soas to generate products 200-300 bp long that would include at least 15bp of the intronic sequence on either side of splice junctions.

For the conformational analysis of single-stranded DNA, samples wereenzymatically amplified using 80 ng of genomic DNA in a total volume of20 μl containing 50 mM KCl, 5 mM Tris-HCl (pH 8.3), 0.01% gelatin, 1.5mmol MgCl₂, 125 μdNTPs, 20 pmol of each unlabeled primer, 0.5 U of Taqpolymerase and 0.15 μl of [α-³² p] dCTP (3000Ci/ml).

Electrophoresis of DNA Fragments Under Nondenaturing Conditions

PCR products were diluted five-fold in a solution containing 95%formamide, 20 mM EDTA, 0.05% bromophenol blue, and 0.05% xylene cyanol.After denaturation at 90° C. for 4 min the samples were placed on ice,and 1.5 μl aliquots were loaded onto 5% nondenaturing polyacrylamidegels (49:1 polyacrylamide:methylene-bis acrylamide) containing 0.5×TBE(1×TBE=90 mM Trisborate, pH 7.8, 2 mM EDTA) (Orita et al., 1989). Eachset of samples was electrophoresed under at least three conditions: a10% glycerol gel at room temperature and at 4° C., and a gel withoutglycerol at 4° C. For the first two conditions, electrophoresis wascarried out at 500 Volts, constant voltage, for 14-20 hours; for thethird, electrophoresis was performed at 15 W, constant power, for 4-5hours. The gels were dried and autoradiographed with an intensifyingscreen for 6-12 hours.

Direct Sequencing of Electrophoretic Variants

Individual bands that presented mobility shifts with respect to wildtype were sequenced as described by Hata et al. (1990), with somemodifications. Each band was excised from the dried gel, suspended in100 μl H2O, and incubated at 37° C. for 1 hr. A 2-μl aliquot wassubjected to enzymatic amplification in a 100-μl reaction volume, withspecific primers augmented at their 5' ends with motifs corresponding touniversal and reverse M13 sequencing primers. The double-strandedproduct resulting from this amplification was isolated byelectrophoresis on a low-melting agarose gel and purified usingGeneClean (Bio 101, La Jolla, Calif.). A second round of enzymaticamplification was usually performed under similar conditions, usingreduced amounts of primers (5 picomol) and of dNTPs (50 μM), and theamplified product was spin-dialyzed with a Centricon 100 column (Amicon,Beverly, Mass.)). Direct sequencing of double-stranded DNA was performedon an ABI 373A DNA sequencer, using fluorescent M13 primers, Taqpolymerase and a thermocycling protocol supplied by the manufacturer(Applied Biosystems, Foster City, Calif.).

Allele-specific Oligonucleotide Hybridization

To verify the presence of molecular variants identified by directsequencing and to determine genotypes, oligonucleotide-specifichybridization was performed. After enzymatic amplification of genomicDNA, each product was denatured with 0.4 N NaOH for 5 min, then spottedin duplicate on nylon membranes (Hybond+, Amersham, Arlington Heights,Ill.), neutralized with 3M Na acetate and cross-linked with UV light.Each membrane was thereafter hybridized with ³² P-end labeledoligonucleotide probes corresponding to wild-type and mutant sequences.After hybridization in 7% polyethylene glycol, 10% SDS, 5 QmM sodiumphosphate, pH 7.0, for 6 hours, the membranes were washed in 6×SSC, 0.1%SDS with a stringency corresponding to the calculated meltingtemperature of the probe. Six molecular variants were subjected to sucha procedure (variants 3, 5, 7, 9, 10, 15 in Table 2). Variant 14 (L359M)was analyzed by the presence or absence of a PstI site (Kunapuli andKumar, 1986) in 140 Utah controls and in the 36 more severelyhypertensive index cases from Utah.

D. Linkage Disequilibrium Between Marker and Variants of AGT

The haplotype distribution of GT alleles and of the variants observed atresidues 174 and 235 were evaluated by maximum likelihood. The M235allele was in strong linkage disequilibrium with the most common GTallele (16 repeats; GT16) while the M235T variant was found incombination with a wide range of GT alleles. The association betweenM235 and GT16 was consistent with the greater frequency of GT16 incontrols than in hypertensives noted earlier. Because the M235T variantoccurred in association with a variety of GT alleles, a greaterfrequency of M235T in cases would not induce spurious genetic linkagebetween hypertension and the GT marker.

E. Assay of Angiotensinogen

Plasma angiotensinogen was measured as the generation of angiotensin Iafter addition of semi-purified human renin to obtain complete cleavageto angiotensin I; the amount of angiotensin I released was measured byradioimmunoassay and angiotensinogen was expressed in ng A-I/ml (Plouinet al., 1989).

EXAMPLE 5 Linkage Analysis Between Renin and Hypertension

The analysis of linkage between renin, the primary candidate, andhypertension was carried out using the methods described in Example 2.

A. RFLP Alleles and Haplotype Frequencies

Similar RFLP frequencies were observed in the 57 hypertensive sib pairprobands and the hypertensive reference group was first verified. AllRFLPs were in Hardy-Weinberg equilibrium and similar proportions werefound in the two groups. Thus, the same haplotype frequencies werededuced from these three RFLPs with eight possible haplotypes and 70%heterozygosity. The six more frequent haplotypes were observed in the133 hypertensive siblings.

B. Observed and Expected Concordances According to Each Sibship Size

The 98 hypertensive sib pairs shared 141 ibs alleles (mean±1standarddeviation=1.44±0.60), while 133.4 (1.36±0.60) were expected under thehypothesis of no linkage, corresponding to a mean excess of 0.08 allelewith a 95% confidence interval of -0.04 to +0.20.

According to each sibship size, 63, 49, and 26 alleles were shared bythe 41 pairs, 13 trios (39 pairs), and 3 quartets (18 pairs),respectively. The corresponding mean observed Z concordances were 0.77,1.89, and 4.33. The comparison of the observed and expectedconcordances, computed in a unilateral t statistic, was not significant(t=0.51, P=0.30).

C. Weights According to the Sibship Sizes

There was a significant excess of ibs allele sharing (13%) when only the41 sib pairs were considered (63 observed vs. 55.8 expected alleles,t=1.93, P<0.03). However, this was negated by the inclusion of the 13trios with 4 alleles less than expected, and of the 3 quartets with anexcess of only 1.5 alleles.

These variations are reflected by the different levels of the t valueaccording to the different weights that take into account the sibshipsize. While the t of 0.52 was computed with w₁ =1, the use of w₂ and W₃,decreasing the weight given to the large sibships, increased the tstatistic although it remained nonsignificant: t₂ =1.34, P=0.09 and t₃=1.16, P=0.12.

D. Discussion

Ninety-eight hypertensive sib pairs from 57 independent sibships wereanalyzed. The hypertensive sibs were selected if they had a strongpredisposition to familial hypertension (at least one parent and onesibling), an early onset of the disease (40.7±12 years), and establishedessential hypertension. Three different RFLPs located throughout therenin gene (TaqI, HindIII, HinfI) were used as genetic markers. Thecombination of these three RFLPs allowed the definition of eighthaplotypes of which six were observed. The allelic frequencies had beenpreviously determined by the analysis of 102 hypertensive subjects(Soubrier et al. 1990) and were confirmed in the 57 hypertensive sibprobands. Taking into account the incomplete heterozygosity of thisrenin marker (70%) and the absence of parental information in 40 of the57 sibships, the alleles shared by the affected sibs were considered asidentical by state and the appropriate statistical test was used (Lange1986). No statistically significant difference was found between theobserved frequencies of total, half, or null allelic concordances andthose expected under the hypothesis of no linkage between the renin geneand hypertension. When the pairs were analyzed independently, theseproportions were of 0.50 vs. 0.45, 0.43 vs 0.48, and 0.07 vs 0.07 forthe observed vs. expected values, respectively, giving a chi-square (2df)=1.21, which was not significant. The most appropriate statistic,using the mean number of marker alleles shared by the sibs (Blackwelderand Elston, 1985) and adding the information obtained in each familyaccording to the affected sibship size, did not demonstrate significance(t=0.51, P=0.30), with only a 5.7% excess of i.b.s. renin alleles sharedby the 98 hypertensive sib pairs. When the reciprocal of the square rootof the variance of the concordance index for each sibship size was usedto maximize the power of the test (Motor and Thomson 1985), the t valueincreased (t=1.31) but remained nonsignificant (P=0.09). Thus, noassociation was found between renin and hypertension.

EXAMPLE 6 Linkage Analysis Between ACE and Hypertension

The analysis of linkage between ACE and hypertension was carried outusing the methods described in Example 3.

A. ACE Growth I-Hormone Linkage

As sib pair linkage tests depend critically on high heterozygosity atthe marker locus (Bishop and Williamson, 1990), cosmids spanning the ACElocus were cloned but failed to identify an informative simple sequencerepeat (data not shown). Since the ACE gene has been localized by insitu hybridization to 17q23 (Mattei et al., 1989) a geneticallywell-characterized chromosomal region (Nakamura et al., 1988), the ACElocus was placed on the genetic map by linkage analysis in 35 CEPHpedigrees using a diallelic polymorphism. (Rigat et al., 1990; Righat etal., 1992). Analysis demonstrated strong linkage to markers fLB17.14,pCMM86 and PM8. Multilocus analysis localized the ACE locus betweenpCMM86 and PM8 (odds ratio favoring location in this interval=2000:1).The hGH gene, localized by in situ hybridization to the same region(Harper et al., 1982), has also shown strong linkage to these markers(Ptacek et al., 1991). Its sequence (Chen et al., 1989) enabled thedevelopment of a highly polymorphic marker based on AAAG and AG repeatslying between the eighteenth and nineteenth Alu repetitive sequences ofthis locus. The hGH-A1819 marker displayed 24 alleles and heterozygosityof 94.6% in 132 unrelated subjects. A similar hGH marker has beenreported to show 82% heterozygosity in 22 unrelated subjects(Polymeropoulos et al., 1991). Pairwise linkage analysis using thismarker in 11 CEPH pedigrees demonstrated complete linkage of the hGFIand ACE loci in 109 meioses (log of the odds (lod) score=11.68).Multilocus analysis confirmed complete linkage between the ACE and hGHloci with a 95% confidence interval for recombination between these lociof ±0.02. This tight linkage permits use of the hGH marker as asurrogate for the ACE locus in linkage analysis with little or no lossof power.

B. Sib Pair Analysis

The characteristics of hypertensive pedigrees ascertained in Utah havebeen previously described (see Example 1). All sibs analyzed werediagnosed by hypertensive before 60 years of age (mean 39.3±9.6 yr) andwere on antihypertensive medication. Allele frequencies at ACE and hGHloci were compared between 132 controls (Utah grandparents belonging tothe CEPH reference families) and 149 hypertensive pedigrees). Thefrequencies of the two ACE alleles were similar in the two groups(frequencies of the larger allele were 0.455 and 0.448, respectively),as were the frequencies of the 24 alleles at the hGFI locus, indicatingno linkage disequilibrium between the marker loci and hypertension. Fromthe 149 hypertensive sibships, 237 sib pairs with the hGH marker weregenotyped. In the absence of parental genotypes, allele sharing betweensibs was scored as `identity by state` (i.b.s.) (Lang, 1986). Theexpected number of alleles shared in the total sample under the nullhypothesis of no linkage of the marker locus and predisposition tohypertension as 254.8 (1.075 per sib pair); the observed number ofalleles shared, 255, coincided with this expectation (t=0.01, ns). Thehigh polymorphism of the hGH marker and the large number of sib pairsstudied gives this analysis 80% power to detect a 10.36% excess in thenumber of alleles shared i.b.s., corresponding to a 12.02 or 13.06%excess of alleles `identical by descent` (i.b.d.) under a recessive or adominant model, respectively.

C. Hypertensive Subgroups

The power of such an analysis can be increased by stratifying anaetiologically heterogeneous population into more homogeneous subgroups.Six different subsets of hypertensive pairs were consideredsequentially. As a possible enrichment of the genetic componentdetermining high blood pressure, two subsets were selected: (1) 52 pairsin which both sibs had early onset of hypertension (prior to 40 years ofage); (2) 31 sib pairs with more severe hypertension, in whom two ormore medications were required for blood pressure control. No excessallele sharing was observed in either group. As a control for thepotential influence of obesity, a significant confounding factor, weseparately analyzed the 71 lean hypertensive pairs in which both sibshad a body mass index less than 20 kg m⁻² (mean 25.9±2.3 kg m⁻²). Again,allele sharing did not depart from that expected under randomsegregation of the marker and hypertension.

It is of further interest to stratify for intermediate phenotypes whichcould be related to either the ACE or hGH loci. ACE plasma concentrationshows evidence for a major gene effect but no relation to blood pressurein healthy subjects (Righat et al., 1990; Alhenc-Gelas et al., 1991).Chronic elevations of hGH can induce not only increased lean body massand hypertension but also insulin resistance (Bratusch-Marrain et al.,1982), a common feature in both human hypertension (Ferrannini et al.,1987; Pollare et al., 1990) and SHR (Reaven et al., 1991). Sib pairswith (a) high lean body mass, (b) high fasting insulin levels and (c)high fasting insulin levels after adjustment for body mass, since bodymass is strongly correlated with insulin levels (r=0.40, p<0.001 in thisstudy) were stratified. Again, no departure from random expectation wasobserved in any subgroup.

D. Discussion

These results demonstrate an absence of linkage between the ACE/hGH lociand hypertension in this population. This study had substantial power todetect linkage, analyzing a large number of hypertensive sib pairs andusing in extremely polymorphic marker that displays no recombinationwith ACE. The lack of departure from random segregation of the markerlocus and hypertension, together with the absence of linkagedisequilibrium between ACE and hGH markers and hypertension, exclude thehypothesis that common variants at this locus could have a significanteffect on blood pressure. The analyses of more homogeneous subsets ofhypertensive pairs potentially enriched for a genetic component werealso negative, though the 95% confidence limits on those subject remainlarge. These results do not rule out the possibility that rare mutationof the ACE gene could, like LDL-receptor mutations inhypercholesterolemia (Goldstein and Brown, 1979), have a significanteffect on the trait but account for only a small percentage of affectedindividuals in the population. Thus, no association was found betweenACE and hypertension.

EXAMPLE 7 Linkage Analysis Between AGT and Hypertension

The analysis of linkage between AGT and hypertension was carried outusing the methods described in Example 4. Three distinct steps wereutilized in the analytical approach to identify and confirm a linkagebetween the ACT gene and hypertension: (1) a genetic linkage study; (2)an identification of molecular variants of AGT followed by a comparisonof their frequencies in hypertensive cases and controls; and (3) ananalysis of variance of plasma angiotensinogen concentration inhypertensive subjects as a function of AGT genotypes.

When parental alleles at a marker locus can be identified unambiguouslyin their offspring, the observed proportion of sibling pairs sharing 0,1 or 2 alleles identical by descent (i.b.d.) can be directly compared tothe expected proportions of 1/4, 1/2, and 1/4 under the hypothesis of nogenetic linkage. For a disease of late onset, however, parents areusually not available for sampling. Furthermore, even for a marker withmultiple alleles and high heterozygosity, the identity by state (i.b.s.)of two alleles in a pair of siblings does not imply that they areidentical by descent, that is, inherited from the same parental gene:this allele may have been present in more than one of the four parentalgenes. In such cases, one must express the probability that two allelesin the off-spring be identical by state as a function of mendeliantransmission rules and allelic frequencies in the reference population.The mean number of alleles shared by siblings is then compared to thevalue expected under the assumption of independent segregation ofhypertension and marker through a one-sided Student t-test (Blackwelderand Elston, 1985; Lange, 1986).

After molecular variants of AGT were identified, their frequencies incases and controls were directly compared. The subject in eachhypertensive sibship with lowest identification number was selected asthe index case; the panel of control subjects consisted of a sample ofhealthy, unrelated individuals from the same population.

Lastly, the effect of AGT genotypes on plasma angiotensinogen was testedby analysis of variance of all hypertensive subjects for which ameasurement was available, taking into account gender or population oforigin as an independent, fixed effect.

A. Genetic Linkage Between AGT and Essential Hypertension

A total of 215 sibships were collected at two centers under separatesampling procedures. The Salt Lake City sample consisted of 132sibships, each with at least two hypertensive siblings onantihypertensive medication, which had been ascertained directly fromthe local population. In Paris, patients from 83 families had beenselected in a hypertension clinic on the basis of strict criteria withrespect to blood pressure and body-mass index. The impact of thedifference in ascertainment protocols is reflected in the summarystatistics presented in Table 3. A highly informative genetic marker atthe AGT locus, based on a variable tandem repeat of the sequence motif`GT` (Kotelevstev et al., 1991), was characterized in all studysubjects; reference frequencies and genotypes (FIG. 1) were determined.Because of the anticipated etiological heterogeneity of this disease,analyses were performed not only on total samples, but also onpre-defined subsets of the data which had the potential of exhibitinggreater genetic homogeneity, such as subjects with earlier onset or withmore severe hypertension (Jeunemaitre et al., 1992). Linkage did notreach significance in the total sample from Salt Lake City (t=1.22,p=0.11). However, a 7.7% excess of alleles shared by hypertensivesiblings was observed in the total sample from Paris (t=1.71, p<0.05),and a slightly greater level of significance was achieved when bothsamples were pooled (t=2.02, p=0.02, Table 4). Similar results wereobserved when only subjects with earlier onset of hypertension wereconsidered (Table 5). By contrast, a more significant, 15% to 18% excessof alleles shared by the sibling pairs was observed when analysis wasrestricted to patients with "more severe" hypertension, predefined inboth groups as subjects requiring two medications for blood pressurecontrol or with diastolic blood pressure equal to or greater than 100mmHg (Table 5). In addition to the greater significance achieved bypooling the "more severe" hypertensive pairs from both studies (t=3.40,p<0.001), the replication of this finding in two different hypertensivepopulations is of critical relevance in evaluating this statisticalevidence.

                  TABLE 4                                                         ______________________________________                                        Sib Pair Linkage Analysis at the Angiotensinogen                                Locus in Salt Lake City and Paris                                                  Sibships                                                                              Pairs  Alleles Shared   Significance                           Study  n       n      Observed/Expected                                                                        Excess                                                                              t    p                                 ______________________________________                                        Salt Lake                                                                       City                                                                          Pairs 102 102 132/126.9                                                       Trios  20  60 85/74.7                                                         Quartets  7  42 56/52.3                                                       Quintet  1  10  8/12.4                                                        Sextets  2  32 31/37.3                                                        TOTAL 132 244 312/303.6 3.8% 1.22 0.11                                        Paris                                                                         Pairs  62  62 86/74.4                                                         Trios  19  57 71/70.7                                                         Quartet  1  6 9/7.5                                                           Quintet  1  10 8/10                                                           TOTAL  83 136 175/162.6 7.7% 1.71 <.05                                        TOTAL 215 379 487/466.2 5.1% 2.02 0.02                                      ______________________________________                                         For each sample, the following is reported; the number of sibships and        sibling pairs analyzed, the observed and expected number of alleles share     by the siblings for each sibship size and the excess of alleles shared (%     after weighting by sibship size.                                         

                  TABLE 5                                                         ______________________________________                                        Genetic Linkage in Sib Pairs Selected                                           for Early Onset or More Severe Hypertension                                                   Alleles Shared                                                Pairs (observed/                                                              n expected) Excess t p                                                      ______________________________________                                        Age Dx < 45 years                                                               Salt Lake City 110 143/136.9  7.1% 1.51 p = 0.07                              Paris  61 80/71.6 11.6% 1.68 p < 0.05                                         TOTAL 171 223/208.5  8.6% 2.23 p < 0.02                                       Rx ≧ 2 drugs or                                                        DBP ≧ 100 mmHg                                                         Salt Lake City  50 74/62.3 18.0% 2.58 p < 0.01                                Paris  60 85/72.9 15.3% 2.25 p < 0.02                                         TOTAL 110 159/136.2 17.1% 3.40 p < 0.001                                    ______________________________________                                    

Because estrogens stimulate angiotensinogen production (Cain et al.,1971; Menard and Catt, 1973), the data were partitioned by gender (Table6). In the Salt Lake City as well as in the Paris samples, linkageremained significant among male-male pairs only (t=2.42, p<0.01, samplespooled). Furthermore, the 37 male-male pairs from both samples who alsomet the criteria for `more severe` hypertension exhibited a 33% excessof shared alleles (t=3.60, p<0.001). Forty-eight women in the Salt LakeCity sample were taking synthetic estrogens or oral preparationscontaining natural estrogens, while none in the Paris sample were doingso; still, there was no excess of shared alleles among the 35 Utahfemale pairs who were not taking exogenous estrogens.

                  TABLE 6                                                         ______________________________________                                        Genetic Linkage in Hypertensive Sib Pairs of Same Gender                                        Alleles Shared                                                Pairs (observed/                                                              n expected) Excess t p                                                      ______________________________________                                        Male-Male Pairs                                                                 Salt Lake City 60 81/74.6 11.0% 1.70 p < 0.05                                 Paris 37 52/44.4 15.4% 1.76 p < 0.05                                          TOTAL 97 133/118.0 12.7% 2.42 P < 0.01                                        Female-Female                                                                 Pairs                                                                         Salt Lake City 79 96/98.3 -2.3% <0                                            Paris 36 45/43.7   1.4% 0.31 p = 0.38                                         TOTAL 115  139/142.0 -1.2% <0                                               ______________________________________                                    

B. Association Between Hypertension and Molecular Variants of AGT

The observation of significant genetic linkage between essentialhypertension and a marker at the AGT locus suggested that molecularvariants in this gene might be causally implicated in the pathogenesisof essential hypertension. A direct search for such variants in allexons and in a 682-bp segment of the 5' noncoding region of AGT wasperformed on a sample consisting of the index cases of the more severelyhypertensive pairs from both populations. Variants detected byelectrophoresis of enzymatically amplified DNA segments undernondenaturing conditions (Orita, 1989) were submitted to direct DNAsequencing (Hata et al., 1990) (FIG. 2). At least 15 distinct molecularvariants have been identified, including five nucleotide substitutionsin the 5' region of the gene, and ten silent and missense variants (FIG.3, Table 2). No variants have been detected within the N-terminalportion of exon 2 that encodes the site cleaved by renin.

The prevalence of each identified variant was compared betweenhypertensive index cases and control subjects. For the Salt Lake Citysample, the first variant detected, M235T (a change from methionine tothreonine at amino acid 235 of AGT), was significantly more frequent inall hypertensive index cases than in controls, with a further increasein frequency among the more severely affected index cases (Table 7).These results were replicated in the Paris sample. The association wassignificant in either sex. In particular, M235T was significantly moreprevalent among female hypertensives (0.51) than in controls (0.37) (χ²₁ =16.9, p<0.001).

                                      TABLE 7                                     __________________________________________________________________________    Linkage Disequilibrium Between Controls and Hypertensives                                    T174M       M235T                                                        n    q    X.sup.2.sub.1                                                                        q    X.sup.2.sub.1                                 __________________________________________________________________________    Salt Lake City                                                                  Controls 280 .08  .35                                                         All Probands 264 .12 2.8, ns .44 4.5, p < 0.05                                More Severe Probands  72 .19 8.4, p < 0.01 .49 4.5, p < 0.05                  Paris                                                                         Controls 184 .09  .38                                                         All Probands 166 .18 5.9, p < 0.02 .52 6.7, p < 0.01                          More Severe Probands  88 .19 5.5, p < 0.02 .52 4.2, p < 0.05                  Total                                                                         Controls 464 .09  .36                                                         All Probands 430 .14 5.3, p < 0.05 .47 11.1, p < 0.001                        (males/females) (224/206) (.16/.13)  (.44/.51)                                More Severe Probands 160 .17 7.4, p < 0.01 .51 11.6, p < 0.001                (males/females) (96/64) (.14/.21)  (.45/.59)                                __________________________________________________________________________     In each group, are indicated the number of alleles analyzed (n), the          allele frequency (q), and the significance of the association between         controls and hypertensives calculated with a Chi square 1 d.f. All            probands refers to the index hypertensive subjects of each sibship (n =       132, Salt Lake City; n = 83, Paris); more severe probands refers to the       index subjects of the more severely affected pairs. There was no              significant difference in  # M235T and T174M allelic frequencies between      males and females. No departure from HardyWeinberg equilibrium was            observed in repartition of these genotypes.                              

Of the other variants tested, only T174M also displayed significantassociation in both samples (Table 7). Analysis of the distribution ofM235T and T174M genotypes indicates that these two variants were incomplete linkage disequilibrium (χ² ₄ =36.4, p<0.0001): T174M waspresent in a subset of chromosomes carrying the M235T allele. When thefrequencies of these haplotypes were contrasted among hypertensives andcontrol subjects, haplotypes carrying M235T, with or without T174M, wereobserved more often among all hypertensive index cases (0.14 and 0.33,n=215) than in controls (0.09 and 0.28, n=232), both differences beingsignificant (χ² ₁ =5.6, p<0.02 and χ² ₁ =13.5, p<0.01).

C. Association with Plasma Concentrations of Angiotensinogen

A possible relationship between plasma concentrations of angiotensinogenand two molecular variants of this protein (M235T and T174M) was testedby analysis of variance, as a function of genotype and gender, inhypertensive subjects in each sample. Women taking oral preparations ofestrogens were excluded from this analysis. No significant differenceswere observed when subjects were classified according to genotype atresidue 174. By contrast, plasma concentrations of angiotensinogen weresignificantly higher in women carrying the M235T variant in eachpopulation sample; when both samples were jointly considered in ananalysis of variance taking into account gender and population as fixedeffects, genotypic differences were highly significant (F₂,313 =14.9,p<0.0001) (Table 8).

                                      TABLE 8                                     __________________________________________________________________________    Influence of the M2325T Variant on Plasma Angiotensinogen Concentrations      M235T  AA       Aa        aa         Significance: F,p                        __________________________________________________________________________    Salt Lake City                                                                       1422 ± 247                                                                       (67)                                                                             1479 ± 311                                                                       (109)                                                                             1641 ± 407                                                                       (33)  5.92, p < 0.005.sup.1                     Males 1376 ± 247 (42) 1404 ± 265 (59) 1499 ± 207 (18)  1.53,                                            ns                                         Females 1500 ± 232 (25) 1566 ± 340 (50) 1811 ± 519 .sup.                                                (15).sup.2,3  3.91, p < 0.02                                                   Paris 1085 ± 210 (32) 1318 ±                                           383 (55) 1514 ± 511 (29)  7.90, p                                          < 0.001.sup.1                              Males 1086 ± 244 (17) 1311 ± 290 .sup. (26).sup.4 1377 ± 606                                            .sup. (10).sup.2  2.82, p = 0.07                                               Females 1084 ± 173 (15) 1324                                              ± 456 .sup. (29).sup.4 1586 ±                                           455 .sup.  (19).sup.2,3  6.44, p <                                            0.01                                       Total 1313 ± 283 (99) 1425 ± 344 (164)  1582 ± 459 (62) 14.90,                                          p < 0.0001.sup.5                           Males 1293 ± 277 (59) 1375 ± 274 (85) 1456 ± 391 .sup.                                                  (28).sup.2  3.10, p < 0.05                 Females 1344 ± 292 (40) 1477 ± 401 (79) 1685 ± 490 .sup.                                                (34).sup.2,3  6.82, p < 0.001            __________________________________________________________________________     Plasma angiotensinogen concentrations are expressed as mean ± 1 S.D.       (ng/ml).                                                                      A: allele M235;                                                               a: allele 235T.                                                               The statistical significance is tested by:                                    (1) oneway analysis of variance;                                              (2) twoway analysis of variance with gender as a fixed effect.sup.1 ; and     (3) threeway analysis of variance with gender and population as fixed         effects..sup.5                                                                .sup.2 p < 0.05 between heterozygotes and homozygotes M235.                   .sup.3 p < 0.05 between homozygotes M235T and homozygotes M235.               .sup.4 p < 0.05 between homozygotes M235T and heterozygotes.             

The effect associated with M235T appeared to be codominant in females.Higher concentrations were found in females than males in Salt Lake City(t=4.3, p<0.001) but not in Paris (t=1.41, p=0.16). While the effect ofestrogens on angiotensinogen production may account for the genderdifference noted in Salt Lake City, a difference in mean values betweenthe two samples is less likely to be of physiological significance; allsubjects belonging to a given population sample were assayedconcurrently and referred to the same standard, but measurements forSalt Lake City and Paris samples were performed 6 months apart, usingdifferent preparations of renin and different standards.

D. Analysis of the G-6A Variant

The data detailed above contained insufficient evidence concerning theG-6A variant which led to the interpretation that the G-6A variantoccurred only in total association with A-20C variant. Thus, in Table 2,it was reported that the frequency of the genes carrying both A-20C andG-6A variants was 14% in Utah Controls (C) and 19% in Utah Hypertensives(H). The frequency was 18% in French hypertensives, with no restingperformed in French controls. The frequency of the M235T variant was 36%and 38% in Utah and French controls, respectively. While this data iscorrect, it did not take into consideration the fact that the G-6Avariant could also occur on genes which did not carry the A-20C variant.

Studies were conducted in Japanese and Utah populations and analyzed asdescribed above. In tests involving 107 random Caucasian controls fromUtah and 99 random Japanese individuals, the G-6A variant was seen inover 90% of the Caucasian genes carrying M235T and in 98% of thecorresponding Japanese genes. The G-6A variant was seen in only oneCaucasian gene carrying the native M235 gene. See Table 9. Thus, morethan 90% of the Utah hypertensives carrying M235T also carry G-6A,rather than the 40% suggested in Table 2.

                  TABLE 9                                                         ______________________________________                                        Frequencies of AGT Haplotypes in Caucasian and Japanese Controls                   Haplotype      Caucasians                                                                              Japanese                                        ______________________________________                                        M235.sup.1, G(-6).sup.1                                                                       0.607     0.242                                                 M235 , A(-6) 0.005 0.000                                                      T235 , G(-6) 0.038 0.015                                                      T235 , A(-6) 0.350 0.743                                                      Total 1.000 1.000                                                           ______________________________________                                         .sup.1 The wildtype sequence at these positions.                         

Discussion

Three sets of observations--genetic linkage, allelic associations, anddifferences in plasma angiotensinogen concentrations among AGTgenotypes--in two independent samples of hypertensive subjectsestablishes involvement of angiotensinogen in the pathogenesis ofessential hypertension.

1. Genetic Linkage in Hypertensive Siblings

Genetic linkage was inferred through the application of first principlesof Mendelian genetics to pairs of related individuals (Blackwelder andElston, 1985), an approach requiring a large number of affected pairsand a highly polymorphic marker at the test locus (Risch, 1990; Bishopand Williamson, 1990). This study design is well suited to commondisorders where the anticipated multiplicity and heterogeneity of causalfactors defies conventional approaches that rely on explicit formulationof a model of inheritance.

In the Utah sample, significant linkage was achieved only for the subsetof more severely affected subjects--as defined by the use of twoantihypertensive drugs or by a diastolic blood pressure equal to orgreater than 100 mmHg; by contrast, linkage reached significance in thetotal sample in Paris. This observation most likely reflects thedifferent ascertainment schemes applied in each study. Salt Lake Citysibships represent a population-based collection of hypertensivesubjects, whereas subjects in Paris were recruited through referral to ahypertension clinic and with the application of strict exclusioncriteria (see Example 1). The former sample has the merit of beingpopulation-based; however, the inclusion of less severely affectedsubjects, as reflected by lower treated blood pressure values than inthe French sample, may have led to the appearance of greater etiologicalheterogeneity in the total sample.

2. Association Between Hypertension and Molecular Variants of AGT

Genetic linkage indicated that variants of AGT could be involved in thepathogenesis of essential hypertension. Among the 15 molecular variantsof the AGT gene identified, significant association with hypertensionwas observed for two distinct amino acid substitutions, M235T and T174M.The significance of this association was established by contrastingallelic frequencies in hypertensive and control subjects. Although thisdesign is liable to biases due to uncontrolled stratification, threearguments support the interpretation that the observed associations arenot spurious: (1) significance is obtained in independent samples fromtwo different populations; (2) gene frequencies are remarkably similarin these two samples, suggesting that little variation should beanticipated among Caucasians of Northern and Western European descent;(3) no differences in allelic frequencies among these hypertensive andcontrol groups have been observed at other loci including renin,angiotensin converting enzyme and HILA (Examples 5 and 6).

Variants M235T and T174M exhibited complete linkage disequilibrium, asT174M occurred on a subset of the haplotypes carrying the M235T variant,and both haplotypes were observed at higher frequency amonghypertensives. Several interpretations can be proposed to account forthis observation: (1) M235T directly mediates a predisposition tohypertension; (2) an unidentified risk factor is common to bothhaplotypes; (3) each haplotype harbors a distinct risk factor.

Although both variants were found significantly more often in femalehypertensives than in control subjects, no linkage was evident amongpairs of female hypertensives in either sample. These observations couldbe reconciled by postulating that angiotensinogen contributes tohypertensive risk directly in males but indirectly in females, whereanother estrogen-modulated factor may mediate the impact of theangiotensinogen-associated predisposition; documented differences in theeffects of testosterone and estrogens on the regulation of genes of therenin-angiotensin system support this hypothesis (Bachmann et al.,1991). While it is conceivable that the predispositions identified bylinkage and by association represent independent variants, the parallelincrease of both association and linkage in subsets of the data suggeststhat they are two manifestations of the same genetic determinant.

In addition to the significant association with hypertension initiallynoted for M235T and T174M variants, analysis of the G-6A variant showsthis variant is also significantly associated with hypertension. Thevery strong association observed between M235T and G-6A has twoimportant implications: (1) all associations noted between M235T andhypertension extend directly to G-6A, that is, G-6A is also diagnosticof a predisposition to hypertension; (2) it is unlikely that statisticaltests alone can resolve the relative merit of either one as a marker forthis predisposition, as it would require much greater sample sizes thanthose analyzed so far. These new observations now dictate fouralternative interpretations of the data (instead of the former two): (1)M235T and G-6A both serve as markers for a predisposition to thedevelopment of hypertension encoded by yet another, unknown molecularvariant; (2) M1235T is causally involved while G-6A is a passive"hitchhiker"; (3) G-6A is causally involved, M235T being the passive"hitchhiker"; or (4) both M235T and G-6A are causal; each contributes adifference in the function of the angiotensinogen gene and protein.Regardless of which interpretation may be correct, the data clearlydemonstrates the significant association of each variant withhypertension and the ability of both two variants to be an indicator ofpredisposition to hypertension.

In view of these findings, molecular variants of the angiotensinogengene constitute an inherited predisposition to essential hypertension inhumans.

EXAMPLE 8 Screening for ACT Variants

Healthy subjects and pregnant women were screened for the M235T variantusing PCR amplification and allele-specific oligonucleotidehybridization as described in Example 4. It was found that healthysubjects who carried the M235T variant had plasma levels ofangiotensinogen higher than in non-carriers, and also had higher bloodpressure. Both of these differences were found to be statisticallysignificant. It was also found that the variant was not limited toCaucausians. The M235T variant was found to be significantly increasedin women presenting preeclampsia during pregnancy.

While the invention has been disclosed in this patent application byreference to the details of preferred embodiments of the invention, itis to be understood that the disclosure is intended in an illustrativerather than in a limiting sense, as it is contemplated thatmodifications will readily occur to those skilled in the art, within thespirit of the invention and the scope of the appended claims.

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    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 22                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - 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-     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Homo sapi - #ens                                       - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                              - - CACTTGCAAC TCCAGGAAGA CT           - #                  - #                     22                                                                    __________________________________________________________________________

What is claimed is:
 1. A method for determining the predisposition of ahuman to hypertension which comprises analyzing the DNA sequence of theangiotensinogen (AGT) gene of said human for the mutation A-20C, wherebythe presence of said mutation is indicative of a predisposition of saidhuman to hypertension.
 2. The method of claim 1 wherein the genomicsequence of the AGT gene of said human is analyzed.
 3. The method ofclaim 1 wherein the cDNA sequence of the AGT gene of said human isanalyzed.
 4. The method of claim 1 wherein a part of the genomicsequence of the AGT gene of said human is analyzed.
 5. The method ofclaim 1 wherein a part of the cDNA sequence of the AGT gene of saidhuman is analyzed.
 6. The method of claim 1 wherein said analysis iscarried out by hybridization.
 7. The method of claim 6 wherein saidhybridization is with an allele-specific oligonucleotide probe.
 8. Themethod of claim 1 wherein said analysis is carried out by sequenceanalysis.
 9. The method of claim 1 wherein said analysis is carried outby SSCP analysis.